Description:
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Remove-iT?PNGaseFisanamidasewhichcleavesbetweentheinnermostGlcNAcandasparagineresiduesofhighmannose,hybrid,andcomplexoligosaccharidesfromN-linkedglycoproteins(1).Remove-iTPNGaseFistaggedwithachitinbindingdomain(CBD)foreasyremovalfromareactionandissuppliedglycerolfreeforoptimalperformanceinHPLCandMSintensivemethods.
ProductSource
Remove-iTPNGaseFispurifiedfromFlavobacteriummeningosepticum(2).Specificity
ReagentsSupplied
Thefollowingreagentsaresuppliedwiththisproduct:
| Storeat(°C) | Concentration |
DTT | -20 | 10X |
GlycoBuffer2 | -20 | 10X |
Notes:
Todeglycosylateanativeglycoprotein,longerincubationtime,aswellasmoreenzyme,mayberequired.UsingtypicalRNaseBdenaturingconditionswithNEBGlycoproteinDenaturingBuffer,containingSDSandDTT,Remove-iTPNGaseFyieldsahigherconcentrationof500,000U/ml.IfusingRemove-iTPNGaseFundertypicalPNGaseFdenaturingconditions,itisessentialtohaveNP-40inthereactionmixtureasRemove-iTPNGaseFisinhibitedbySDS.Itisnotknownwhythisnon-ionicdetergentcounteractstheSDSinhibitionRemove-iTPNGaseFwillnotcleaveN-linkedglycanscontainingcoreα1-3Fucose.Recommendedstoragetemperatureis4°C,avoidrepeatfreeze-thawcyclesRemovalofRemove-iTPNGaseFfromthedeglycosylationreactioncanbescaleduplinearlywithlargervolumesofchitinmagneticbeads.ChitinMagneticBeadsBindingCapacityis0.4μg/μlofCBD-taggedprotein.Toremove1-5μlofRemove-iTPNGaseFuse50μlofchitinmagneticbeads.